Methods for producing large amounts of recombinant protein are well known. As the recombinant protein industry has developed, the need for various quality control assays has arisen. An example is the need for the quantitation of nucleic acids present in recombinant protein preparations. Current FDA guidelines require that the amount of nucleic acid present in recombinant therapeutic proteins be less than 10 pg of DNA per daily dose of recombinant protein. Therefore, methods for detecting extremely low amounts of nucleic acids are needed. Such methods can also find widespread use for the quantitation of nucleic acid in forensic, clinical and agricultural samples.
Several methods of detecting low levels of nucleic acid have been described. One method is based on classical hybridization techniques. This method utilizes radiolabeled nucleic acid probes which bind to the nucleic acid of interest. However, this method has several disadvantages, including poor reproducibility, generation of large amounts of radioactive waste reagent, and high background levels caused by nonspecific binding. Furthermore, this technique is generally inappropriate for determining the presence of low amounts of nucleic acid of unknown sequence.
A second method of detecting nucleic acid utilizes fluorescent dyes capable of intercalating into nucleic acids. However, many interfering substances such as detergents, proteins, and lipids affect the reproducibility of the signal generated by this method.
A third method of detecting low levels of DNA utilizes biotinylated single-stranded DNA binding protein (SSB), streptavidin, an anti-DNA antibody fused to urease, and biotinylated nitrocellulose as reagents. This assay is commercially available from Molecular Devices (Sunnyvale, Calif.) and described in Kung et al., Anal. Biochem., 187:220-27 (1990). The assay is performed by incubating the streptavidin, biotin-SSB, and the anti-DNA antibody together, permitting a complex to be formed. The complex is then captured on the biotinylated membrane, washed, and the amount of captured urease is determined. This method is highly sensitive but has several disadvantages, including costly reagents and the need for extensive controls.
A fourth method takes advantage of depolymerization by polymerases. Polynucleotide polymerases are responsible for the synthesis of nucleic acids in cells. The reverse of this reaction, the depolymerization of nucleic acid, can also occur in the presence of phosphate (phosphorolysis) or pyrophosphate (pyrophosphorolysis). Enzymes reported to carry out pyrophosphorolysis include E. coli DNA Polymerase (Deutscher and Kornberg, J. Biol. Chem., 244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al., Biochemistry 30:526-37 (1991); Tabor and Richardson, J. Biol. Chem. 265: 8322-28 (1990)), E. coli RNA polymerase (Rozovskaya et al., Biochem. J. 224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak, J. Biol. Chem. 255: 2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al., J. Biol. Chem. 269:24195-202 (1994)).
U.S. Pat. No. 4,735,897 describes a method of detecting polyadenylated messenger RNA (poly(A) mRNA). Depolymerization of poly(A) mRNA in the presence of phosphate has been shown to result in the formation of ADP, which can be converted by pyruvate kinase or creatine phosphokinase into ATP. RNA may also be digested by a ribonuclease to AMP, converted to ADP by adenylate kinase, and then converted to ATP by pyruvate kinase. The ATP so produced is detected by a luciferase detection system. In the presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light which can then be quantitated using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin.
The presence of ATP-generating enzymes in all organisms also allows the use of a luciferase system for detecting the presence or amounts of contaminating cells in a sample, as described in U.S. Pat. No. 5,648,232. For example, ADP may be added to a sample suspected of containing contaminating cells. The ADP is converted by cellular enzymes into ATP which is detected by a luciferase assay, as described above. A major disadvantage of this method is the relative instability of the ADP substrate.
The polymerase chain reaction (PCR) is a well known method for detecting specific nucleic acids. In PCR, two primers are utilized, one that hybridizes to the sense strand of a DNA target and one that hybridizes to the antisense strand of the DNA target. The DNA is denatured by heating to yield single strands and the primers permitted to hybridize to the respective strands. A polymerase and dNTPs are then used to synthesize new DNA strands based on the sequence of the target strands and extended from the primers. Repeated cycles result in the amplification of a DNA product bounded at its 5' and 3' ends by the two primers. PCR is extremely sensitive, but contamination from previously amplified product can limit its usefulness in clinical applications. Also, it is of limited use for the detection of nucleic acid of unknown sequence.
What is needed in the art are reliable, cost-effective methods of detecting extremely low levels of nucleic acids, specific nucleic acids, cells, and cellular material in a wide variety of samples.